novel application of fibrinogen-420 and its active domain

ABSTRACT

The invention discloses a novel application of fibrinogen-420 and its active domain (alpha EC domain), and a separate alpha EC domain protein has the same or similar function with fibrinogen-420. Fibrinogen-420 and its active domain can be widely used in inhibiting protein aggregation, helping protein refolding, drugs which can prevent and/or treat protein conformation disease, detecting denatured protein in quality control and protect protein from denaturation.

FIELD OF THE INVENTION

This invention relates to a novel application of Fibrinogen-420 and itsactive domain.

BACKGROUND OF THE INVENTION

Fibrinogen, also known as coagulation factor I, is an important proteinin the process of blood clotting. Fibrinogen has a molecular weight of340,000 Daltons, and is composed of two subunits connected by disulfidebonds to form dimers. Each subunit respectively consists of threeintertwined polypeptide chains, called A, B, C chain. In the process ofclotting, fibrinogen digested by thrombin to generate fibrin and thusform insoluble fibrin polymers. And then the composition of fibrinpolymer fibers and blood platelets will form solid tampon. Fibrinogen isalso a stress protein, whose content in the blood is about 1.5-4 mg/ml.The content of fibrinogen is related to the immune status, which couldalso reflect the risk of cardiovascular disease.

Fibrinogen-420 is a subtype of fibrinogen, in which the C-terminal of Achain has an extension of globular domain than the ordinary fibrinogen.This extension of globular domain is called the alpha EC domain, whichhas high homology with the globular domain at the end of B, C chains.The molecular weight of fibrinogen-420 is about 420,000 Daltons, whichis different from the general tissue fibrinogen of 340,000 Dalton.

Protein misfolding disease is due to the conformational change ofspecific proteins in the tissue, during which proteins gather to produceamyloidosis and finally result in a class of diseases with pathologicalchanges in tissues and organs, such as Alzheimer's disease and bovinespongiform encephalopathy. There have been no effective preventivetreatments or therapeutics for these diseases by now. The existingmethods such as monoclonal antibody technology, small molecules,synthetic peptides, et al. have many disadvantages including immunerejection reactions, lacking of broad-spectrum, significant sideeffects, and a short half-life in vivo.

There are a large number of heat shock proteins or chaperones, protectcells from high temperature, free radicals, organic solvents (egethanol) and other damages when the body suffers from stimulation. Butthe heat shock proteins do not exist in the circulatory system. Themechanism by which the body protects extracellular protein is unclear.

DESCRIPTION OF THE INVENTION

The purpose of this invention is to provide novel applications ofFibrinogen-420 and its active domain.

Our study shows that fibrinogen-420 has molecular chaperone activity andbroad-spectrum, non-specific protective effects. Fibrinogen-420 is anendogenous protein, so that it will not be quickly degraded in the bodyand does not arouse immune rejection. It can promote denatured proteinsto refold properly and stabilize protein conformation and function. Soit can be widely used in protein refolding, denatured protein testing inquality control, and prevention of protein denaturation et al. Theprotein described could be a recombinant protein or natural protein.

Fibrinogen-420 contains alpha EC domain, and a separated alpha EC domainprotein has the same or similar functions with intact fibrinogen-420.The amino acid sequence of alpha EC domain is shown as SEQ ID NO. 1, andthe fibrinogen-420 could specifically refer to human fibrinogen-420.

Fibrinogen-420 or alpha EC domain can be prepared into the proteinreagent for use. Described protein reagents include at leastfibrinogen-420 or alpha EC domain protein, and the protein reagents donot rule out other solvents and additives. Good results are expectedwhen the ratio of other protein vs. fibrinogen-420 or alpha EC domainprotein is in the range of 25:1 to 1:100, among which the ratio of 1:1is included. The best ratio depends on the requirement of specificapplication.

The present invention also shows that fibrinogen-420 or alpha EC domainprotein can inhibit the aggregation of degeneration protein, and protectthe protein activity as well. Thus it can be used as drugs to treatprotein conformation related diseases.

Wherein, the drugs to treat protein misfolding diseases containfibrinogen-420 or alpha EC domain protein as the active ingredients.Therapeutic proteins as described can be used for the treatment of avariety of protein degenerative diseases, such as protein denaturationcaused by fever, tobacco, alcohol, oxygen free radicals and otherharmful substances.

The drugs for the treatment of protein misfolding diseases containfibrinogen-420 or alpha EC domain protein as the active ingredients.

In this invention, fibrinogen-420 or alpha EC domain protein can capturethe protein unfolding process. By helping the protein to refoldcorrectly or keeping it in a folded state, fibrinogen-420 or alpha ECdomain protein can prevent protein aggregation and stabilize theactivity and function of a protein. Thus, fibrinogen-420 or alpha ECdomain protein can enhance the ability of a protein againstdenaturation, so that it can be used as a protein stabilizing agent invitro.

Wherein the protein stabilizing agent described contains fibrinogen-420or alpha EC domain protein as the active ingredient. The proteinstabilizing agent described can inhibit the precipitation of proteinswhich are easy to gather and form aggregation. The protein stabilizingagent can also protect the enzyme activity, such as stabilize citratesynthase, luciferase, insulin and the activity of other enzymes.

In biological diagnostic kits, particularly in ELISA immunoassaydiagnostic kits, fibrinogen-420 or alpha EC domain protein can stabilizethe protein reagents especially the antibodies cross-linked withreporter enzymes (such as horseradish peroxidase, alkaline phosphataseor luciferase), and increase the shelf life and the quality of theproducts.

Fibrinogen-420 or alpha EC domain protein can also be used to identifyunfolding and denatured proteins, so it can be used in the qualitycontrol of protein reagents.

FIGURES

FIG. 1. Fibrinogen-420 or alpha EC domain protein can inhibit thethermal-induced denaturation and aggregation of citrate synthase.

FIG. 2. The alpha EC domain protein inhibits the chemical denaturationand aggregation of citrate synthase.

FIG. 3. Fibrinogen-420 inhibits the thermal-induced denaturation andinactivation of citrate synthase.

FIG. 4. The alpha EC domain protein inhibits the thermal-induceddenaturation and inactivation of citrate synthase.

FIG. 5. The alpha EC domain protein specifically recognizes thedenatured citrate synthase protein.

EXAMPLES Example 1 Fibrinogen-420 and Alpha EC Domain Protein SuppressDenatured Aggregation of Citrate Synthase

a. Preparation of Fibrinogen-420 and Alpha EC Domain Protein

The preparation of fibrinogen-420 started from the purification of afibrinogen mixture from blood or cord blood, after which fibrinogen-420can be further purified. Details are as follows:

(1) Purification of fibrinogen mixture: first, add protease inhibitorsinto fresh blood or cord blood, then centrifuge at 4° C. 2000 rpm andget yellow plasma from the supernatant. Then add glycine dry powder tothe plasma while stirring to make glycine completely dissolved, and thefinal concentration of glycine is 2.1 M. After centrifugation at 5000rpm for 15 min, the white flocculent precipitate is obtained. Dissolvethe precipitate with buffer (0.15 M NaCl, 0.01 M sodium phosphate, pH6.4 solution) that is ⅓ of the original plasma volume, and repeat thisstep until the dissolved volume is 1/10 of the original plasma volume.Add an equal volume of water to dilute. Then put the diluted solution at2-5° C. for 6 hours, remove precipitate after centrifugation, add anequal volume of 0.3 M sodium chloride solution to the supernatant, thenadd 95% ethanol to a final concentration of 8% ethanol (volume ratio),while keep the temperature at −3° C., fully precipitate and theprecipitation after 5000 rpm centrifugation is used as raw materials forthe next step purification of fibrinogen-420.(2) Purification of fibrinogen-420: dissolve fibrinogen precipitateobtained in step (1) in 0.3 M sodium chloride solution and dialysisagainst 0.005 M Tris-phosphate buffer, pH8.6 (molar concentrationcalculated in accordance with phosphate radical). Mono Q HR 10/10 anionexchange column (Pharmacia) is used as the chromatographic column. UsepH step elution to elute the sample, starting with 0.005 MTris-phosphate buffer rapid transition to 0.2 M Tris-phosphate buffer,pH 6.0, and then maintain the 0.2 M Tris-phosphate buffer, pH 6.0 toelute 12 column volumes, and finally eluted using a linear gradient for12 column volumes to 0.5 M Tris-phosphate buffer, pH 4.2. Fibrinogen-420is obtained in the last step of linear elution. The protein can bestored after dialysis against 125 mM sodium chloride, 25 mM HEPES buffer(pH 7.4).

Alpha EC domain protein is obtained as follows:

Alpha EC domain protein refolding and purification: Use the human livercDNA library as a template for PCR amplification. Primers as follows:

5-GGAATTCCATATGGACTGTGATGATGTCCTCC-3′5-ACCGCTCGAGCTATTGGGTCACAAGGGGCC-3′

Restriction sites of NdeI and XhoI are introduced in the primers. Theannealing temperature of PCR amplification is 55° C. Connect the αECfragment to pET-30a expression vector (Novagen Inc.) after digested withrestriction enzyme NdeI and XhoI with the same double-restriction sites.Get the recombinant bacteria after transform the recombinant expressionvector into E. coli competent cells BL21/DE3 (Beijing DingGuoBiotechnology Company). Pick monoclonal recombinant bacteria to 10 ml LB(kanamycin 100 μg/ml), overnight. Then transferred to 1 liter of LBmedium (kanamycin 100 μg/ml). Until the bacilli turbidity of OD600reached 0.8 or so, add 0.5 mM IPTG-induced for 4 hours and collectbacteria by centrifugation. Collect the inclusion body protein afterbreaking cells. Restore the dissolved inclusion body protein, and thenpurify by anion-exchange column. The sample loading buffer as follows:8M urea, 20 mM Tris-HCl and pH 8.0, 30 mM BME; add 1M NaCl to loadingbuffer is elution buffer. Use a linear gradient elution, and collectelution peak step by step. Detect the protein purity by electrophoresis.Select the components of which purity greater than 80% to do therefolding experiments. When refolding, adjust the protein concentrationto less than 0.2 mg/ml with 20 mM Tris-HCl buffer containing 8 M urea(pH 8.0), and then dialyze into 20 mM Tris-HCl, 150 mM NaCl, 1 mMchloride calcium with pH 8.0. Exchange a dialysis fluid interval of atleast 4 hours, and exchange refolding solution at least for two times,fully dialysis overnight. Finally, dialyze to 20 mM Tris-HCl loadingbuffer for the recovery of purified protein. Use anion exchange columnfor purification. The sample should be centrifuged or filter with 0.22micron pore size membrane before add to column. Use a linear gradient ofsalt ions, and collect protein peaks step by step. Detect the purity byoxide gel electrophoresis.

b. Fibrinogen-420 and Alpha EC Domain Protein Inhibit Citrate SynthaseThermal Denaturation Aggregation

Citrate synthase is a key enzyme in the tricarboxylic acid cycle, butits thermal stability is poor. The temperature of 43° C. will make itdenature, aggregate and precipitate. The process of citrate synthaseaggregation can be examined by the changing of light scattering. Themethod is as follows:

Detecting the process of light scattering with florescence instrumentFL4500 (Hitachi, Ltd), adjusting the exciting light, emissionmonochromator to 500 nm and slit width to 2.5 nm Dissolving the citratesynthase in 40 nM HEPES buffer solution and making the finalconcentration to 0.15 μM. Simultaneously, the experiment group 1 isadded with 0.15 μM fibrinogen-420, the experiment group 2 is added with0.15 μM alpha EC domain protein, the control group 1 is added with anequal volume of HEPES buffer solution and the control group 2 is addedwith an equal volume of 1.2 μM bovine serum albumin Putting the samplesinto the 43° C. water bath and detecting the signal of light scattering.Repeat the experiment for 3 times.

The light scattering signal detecting result shown in the FIG. 1indicates that during the process of heating for 200 s the citratesynthase in the control group 1 and 2 begin to aggregate and theintensity of light scattering will be increased. However, citratesynthase aggregation in the experiment group 1 and 2 will be reducedobviously. The effect of inhibition in the experiment group 2 is betterthan that of group 1. Moreover, 0.15 μM alpha EC domain protein canalmost totally inhibit equal molarity of citrate synthase during thethermal denaturation and aggregation process.

In FIG. 1, (∘) represents control group 1, (•) represents control group2, (Δ) represents experiment 1, (□) represents adding 0.6 μM alpha EC.

Example 2 Fibrinogen-420 and Alpha EC Domain Protein Protecting theActivity of Citrate Synthase (CS)

The experimental method of fibrinogen-420 and alpha EC domain proteininhibiting CS inactivation of thermal denaturation is as follows:

Dissolving the citrate synthase in the 40 mM HEPES buffer solution andmaking the final concentration to 0.075 μM. Simultaneously, theexperiment group 1 is added 0.075 μM fibrinogen-420, the experimentgroup 2 is added 0.15 μM fibrinogen-420, the experiment group 3 is added0.15 μM, the experiment group 4 is added 0.15 μM alpha EC domain proteinand the control group is added an equal volume of HEPES buffer solution.Putting the samples into the 43° C. water bath and beginning to detectthe change of the activity of citrate synthase. The activity of citratesynthase is defined 100% before heating.

The method of detecting the activity of citrate synthase is as follows:

930 μL TE buffer solution (50 nM Tris, 2 mM EDTA, pH 8.0), 10 μL 10 mMoxaloacetic acid, 10 μL 10 mM DTNB, 30 μL 5 mM acetyl-CoA. Mixing thesesolutions above, adding them to the solution containing 20 μL citratesynthase quickly and detecting the dynamic change of UV absorption at412 nm wavelength. The linearity curve slope of absorbency changerepresents the activity of enzyme.

The determination result of the activity of citrate synthase shown inthe FIGS. 6 and 7 indicates that with the time going on, the activity ofcitrate synthase in the control group decreases rapidly but allexperiment groups can slow down the activity loss speed effectively.

In FIG. 3, (∘) represents control group, (Δ) represents experiment group1, (□) represents experiment group 2. In FIG. 4, (∘) represents controlgroup, (Δ) represents experiment group 3, (□) represents experimentgroup 4.

Example 3 Alpha EC Domain Protein Recognizing Citrate SynthaseSpecifically

Citrate synthase and alpha EC domain protein are incubated together at43° C. After being heated for 5 min or 10 min, antibodies of citratesynthase and alpha EC domain protein are added into the supernatant toperform co-immunoprecipitation. In the control group, citrate synthaseand alpha EC domain protein are incubated together at room temperatureand antibodies of citrate synthase and alpha EC domain protein are addedinto the supernatant to perform co-immunoprecipitation.

Results shown in the FIG. 5 indicate that after adding the antibody ofcitrate synthase, the denatured citrate synthase can be precipitated andalpha EC domain protein can also be precipitated at the same time. Afteradding the antibody of alpha EC domain protein, both of alpha EC domainprotein and citrate synthase can be precipitated. The results aboveindicate that after being heated, citrate synthase and alpha EC domainprotein can form complex so that the antibody of one protein canprecipitate the other protein at the same time. In the control group,co-immunoprecipitation does not happen. The result illustrates thatalpha EC domain protein can recognize and bind to the thermallydenatured citrate synthase specifically.

In the FIG. 5, above of the figure is the co-immunoprecipitation ofcitrate synthase. Detect with antibody of alpha EC domain protein afterelectrophoresis. Lane 1 is the positive control. Lane 2 shows theco-immunoprecipitation after incubation for 10 min in the roomtemperature. Lane 3 and 4 represent co-immunoprecipitation after beingheated for 5 min and 10 min respectively. Below of the figure is theco-immunoprecipitation with the antibody of alpha EC domain protein,which is detected with antibody of citrate synthase afterecectrophoresis. Lane 1 is the positive control. Lane 2 shows theco-immunoprecipitation after incubation for 10 min. Lane 3 and 4represent co-immunoprecipitation after 5 min and 10 min of incubationrespectively. In the figure, “CS” represents citrate synthase and “alphaEC” represents alpha EC domain protein.

1. The application of inhibiting protein aggregation or refolding thedenatured proteins with the following a) or b) protein; a) Its aminoacid sequence is the sequence 1 in the sequence table; b)Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420. 2.According to the application of claim 1, its characteristic lies in thatthe molar ratio of the described a) or b) protein and denatured proteinis 25:1 to 1:100.
 3. The application in preparation for drugs which canprevent and/or treat protein misfolding diseases with the following a)or b) protein; a) Its amino acid sequence is the sequence 1 in thesequence table; b) Fibrinogen-420; said fibrinogen-420 prefers humanfibrinogen-420.
 4. One drug for preventing and/or treating proteinconformation disease and its active ingredient is the following a) or b)protein; a) Its amino acid sequence is the sequence 1 in the sequencetable; b) Fibrinogen-420; said fibrinogen-420 prefers humanfibrinogen-420.
 5. The application of protein anti-denaturation with thefollowing a) or b) protein; a) Its amino acid sequence is the sequence 1in the sequence table; b) Fibrinogen-420; said fibrinogen-420 prefershuman fibrinogen-420.
 6. One drug for treating protein degenerativediseases and its active ingredient is the following a) or b) protein; a)Its amino acid sequence is the sequence 1 in the sequence table; b)Fibrinogen-420; said fibrinogen-420 prefers human fibrinogen-420.
 7. Oneprotein stabilizing agent whose active ingredient is the following a) orb) protein; a) Its amino acid sequence is the sequence 1 in the sequencetable; b) Fibrinogen-420; said fibrinogen-420 prefers humanfibrinogen-420.
 8. The application in quality control and detection ofprotein products with the following a) or b) protein; a) Its amino acidsequence is the sequence 1 in the sequence table; b) Fibrinogen-420;said fibrinogen-420 prefers human fibrinogen-420.